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RNA-seq
' RNA-Seq' RNA Sequencing, also called "whole Transcriptome Shotgun Sequencing", is a technology that utilizes the capabilities of next-generation sequencing to reveal a snapshot of RNA presence and quantity from a genome at a given moment in time. Key concepts: '(1)transcriptome: the complete set of transcripts ' Technology RNA-Seq uses recently developed deep-sequencing technologies. In general, a population of RNA(total or fractionated, such as poly(A+) is converted to a library of cDNA fragments with adaptors attached to one or both ends. High-throughput sequencing technology can be used for RNA-Seq, and the Illumina IG, Applied Biosystems SOLiD and Roche 454 Life Science systems have already been applied for this purpose. Following sequencing, the resulting reads are either aligned to a reference genome or reference transcripts, or assembled de novo without the genomic sequence to produce a genome-scale transcription map that consists of both the transcriptional structure and/ or level of expression for each gene. Procedure： rRNA removal: vast majority of RNA(>90%) present in cells consists of ribosomal RNA (rRNA). The bulk of the total RNA is not informative as to the true diversity of the transcriptome present in the remaining RNA. In order to avoid wasting effort in re-sequencing the same ribosomal RNA millions of times, several techniques exist to focus the sequencing effort on the non-ribosomal portion. One option is to selectively enrich for mRNA present in the total RNA. This can be accomplished using various commercial kits which either selectively remove rRNA of selectively enrich for mRNA. The rRNA depletion kits(e.g. RiboMinus(Invitrogen)) use antisense versions of the ribosomal transcripts to be removed which are conjugated to either biotinylated or magnetic beads. In the case of polyA enrichment, similar beads that have long oligo dT stretches(e.g. OligoTex(Qiagen)), which bind the polyA tails present on most mRNA molecules, can selectively enrich for non-ribosomal transcripts. 1.Priming: Following enrichment, the resulting mRNA must be primed for the reverse transcription reaction using either random primers or oligo dT primers. The advantate of using oligo dT is that the majority of cDNA produced should be polyadenylated mRNA,hence more of the sequence obtained should be informative(non-ribosomal). 2.cDNA synthesis: In the case of Cloonan, the synthesis of the first cDNA strand from the fragmented RNA is performed using a tagged random hexamer and a modified MMLV Reverse Transcriptase, which allows additional nucleotides to be added to the termini.After the addition of C nucleotides to the 3' ends of the first cDNA strand product, another tagged poly G track primer is used to prime the second strand synthesis, resulting in(PCR amplifiable) double-stranded cDNA molecules with tags that mark the original 5' and 3' ends. 3.Sequencing basic principle: the isolatioon and attachment to a solid matrix of a single DNA fragment through limiting dilution, followed by amplification of this single molecule either through a specialized emulsion PCR(EM_PCR; SOLiD/454) or a linker based bridgeing reaction(Illumina). These larger, discreet populations of identical molecules can then be sequenced in parallel, either through the measurement of the incorporation of fluorescent nucleotides(Illumina) or short fluorescent linkers(SOLiD), or through the release of by-products from incorporation of normal nucleotides. 4. overview of Illumina Genome Analyzer II protocol (1) Fragmentation of the cDNA.(nebulization,or DNase digestion) (2)Purification of the fragmentation products using Qiagen QIA-quick PCR kit or equivalent (3) End repair of cDNA fragments (4) Tailing of cDNA fragments. (5) Adaptor ligation (6)size-based purification of ligation products. (7) PCR of ligation products (8) purification and quantification (9) sequencing of fragments. 5.Data analysis 5.1 Data filtering 5.2 Read mapping 6.Expression scoring and representation Reference： http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2949280/